1. Field of the Invention
The present invention relates to purified nucleic acids encoding Antarctic bacteria (Polaribacter sp.) derived enzymes such as proteinases, which can be a protein, and to purified polypeptides that have high proteolytic activity and belong to the superfamily of subtilisin-like enzymes (subtilases). The present invention also relates to a protein having cold adapted activity, especially specific activity in the range around 4-45° C., and having noticeable activity in the range of 4-20° C. In addition, the present invention relates to a DNA construct comprising a DNA sequence encoding the cold adapted subtilisin-like protease, and a cell including the DNA construct. Furthermore, the present invention relates to a method of preparing the cold adapted subtilisin-like protease by use of recombinant DNA techniques.
2. Description of the Prior Art
The subtilisin-like serine protease (S8) family plays roles in a multitude of diverse bacterial cellular and metabolic processes, such as sporulation and differentiation, protein turnover, maturation of enzymes and hormones and maintenance of the cellular protein pool. Another important function, especially for extracelullar subtilisin-like proteasas, is the hydrolysis of proteins in external cell environments which enables the cell to absorb and utilize hydrolytic products.
Serine proteases are used in numerous and varied industrial contexts and commercial purposes including laundry detergents, food processing, leather processing, medical usage and skin care products. In laundry detergents, the protease is employed to break down organic or poorly soluble compounds to more soluble forms that can be more easily dissolved in detergent and water. Examples of food processing include tenderizing meats, preparation of protein hydrolyzates and maturing cheese. In the case of medical usage, proteases are applied to treat of burns, purulent wounds, furuncles and deep abscesses. Proteases may be included in skin care field to remove scales on the skin surface that build up due to an imbalance in the rate of desquamation.
Common proteases used in some of these applications are derived from prokaryotic or eukaryotic cells that are easily grown for industrial manufacture of their enzymes. For example a common species used is Bacillus as described in U.S. Pat. No. 5,217,878. Alternatively, U.S. Pat. No. 5,278,062 describes serine proteases isolated from a fungus, Tritirachium album, for use in laundry detergent compositions. The advent of recombinant technology allows expression of any species' proteins in a host suitable for industrial manufacturing. The majority of the commercially available proteases used in detergent applications have high optimal temperatures, for example 60° C. Bacteria isolated from cold environments such as Antarctic sea water are psychrophilic microorganisms and are expected to have cold adapted enzymes.
There are some enzymes with cold adapted subtilisin-like activity from psychrophilic microorganisms, for example: Flavobacterium balustinum (Morita, Y., Hasan, Q., Sakaguchi, T., Murakami Y., Yokohama, K., Tamaya, E. (1998) Appl. Microbiol. Biotechnol. 50:669-675), Bacillus TA41 (Davial, S., Feller, G., Narinx, E., Gerday, Ch. (1994) J. Biol. Chem. 269:17448-17453) and Pseudomonas strain DY-A (Zeng, R., Zhang, R., Zhao, J., Lin, N. (2003) Extremophiles 7:335-337). All of these proteins have low stability at ambient temperatures and in the presence of common compounds present in commercial detergents.
Therefore, there is a need for new alternative proteases, in this case subtilisin-like proteases which work at ambient and low temperatures and in the presence of common commercial detergent compositions.